DNA sequencing is one of the cornerstone analytical techniques of modern molecular biology. The development of reliable methods for sequencing has led to great advances in the understanding of genetic information and has made possible the manipulation of genetic material (i.e., genetic engineering).
There are currently two methods for sequencing DNA: the Maxam-Gilbert chemical degradation method [Maxam et al., Meth. in Enzym., Vol. 65, 499-559 (1980)] and the Sanger dideoxy chain termination method [Sanger et al., Proc. Nat. Acad. Sci. USA, Vol. 74, 5463-5467 (1977)]. A common feature of these two techniques is the generation of a set of DNA fragments which are analyzed by electrophoresis. The techniques differ in the methods used to prepare these fragments.
With Sanger's technique, DNA fragments are produced through partial enzymatic copying (i.e., synthesis) of the piece of DNA to be sequenced. In the most common version, the piece of DNA to be sequenced is inserted, using standard techniques, into a "sequencing vector"; a large, circular, single-stranded piece of DNA such as the bacteriophage M13. This becomes the template for the copying process. A short piece of DNA with its sequence complementary to a region of the template just upstream from the insert is annealed to the template to serve as a primer for the synthesis. In the presence of the four natural deoxyribonucleoside triphosphates (dNTP's), a DNA polymerase will extend the primer from the 3'-end to produce a complementary copy of the template in the region of the insert. To produce a complete set of sequencing fragments, four reactions are run in parallel, each containing the four dNTP's along with a single dideoxyribonucleoside triphosphate (ddNTP) terminator, one for each base. (.sup.32 P-labeled dNTP is added to give labeled fragments). If a dNTP is incorporated by the polymerase, chain extension can continue. If the corresponding ddNTP is selected, the chain is terminated. The ratio of ddNTP to dNTP's is adjusted to generate DNA fragments of appropriate lengths. Each of the four reaction mixtures will, thus, contain a distribution of fragments with the same dideoxynucleotide residue at the 3'-terminus and a primer-defined 5'-terminus.
The key to the success of the Sanger method is the ability of the ddNTP's to function as chain terminating substrates which, after incorporation, prohibit further chain extension. The number of available chain terminating substrates is limited. Novel chain terminating substrates would be useful and valuable because they would expand the utility of the Sanger method.
The present invention relates to the use of dideoxyfructonucleotides as chain terminating substrates and deoxyfructonucleotides as chain propagating substrates in DNA chain extension reactions. Specifically, these novel compounds differ from the deoxynucleotides and dideoxynucleotides generally used in extension or termination reactions in that they possess a functional-group-bearing methylene unit at the anomeric carbon of the sugar residue. This one-carbon extension is a potential site for the attachment of reporters (detectable groups) such as fluorescent dyes or biotin. Reporter-labeled chain terminators have been shown to be useful in non-radioisotopic sequencing. Prober et al., Science, 238, 336-341 (1987). Reporter labeled chain propagators are useful in the labeling and detection of DNA fragments. Langer et al., Proc. Natl. Acad Sci. USA, 78, 6633-6637 (1981). In the compounds provided by the instant invention, the reporter may be contained within the sugar portion rather than the purine or pyrimidine portion of the molecule, as in previously developed methodogy. Hence, since the sugar portion of the nucleotide base is common to all nucleotides, a single fructosugar derivative would provide a convenient and universally useful means by which all nucleotides to be used in nucleotide extension or termination reactions could be prepared via a common intermediate.
References which disclose structurally related compounds include: Sturm et al., J. Org. Chem., Vol. 47, 4367-4370 (1982), which discloses a cyclic 4',6'-monophosphate of psicofuranine; Tatsuoka et al., Heterocycles, Vol. 24, No. 8, 2133-2136 (1986), which discloses synthesis of 1'-deoxy-1'-phosphono-1-.beta.-D-fructofuranosyluracil and 1',3'-dideoxy-1'-phosphono-1-.beta.-D-fructofuranosyluracil; Tatsuoka et al., Heterocycles, Vol. 24, No. 3, 617-620 (1986), which discloses synthesis of 2,3'anhydro-1'-phosphono-1-B-D-fructofuranosyl uracil; and Tatsuoka et al., Japanese Patent 61-275290.